Comparative analysis of the nutritional and biological properties between the pileus and stipe of Morchella sextelata

Morchella sextelata is a highly prized edible mushroom and is widely consumed for its distinctive taste and texture. The stipe of M. sextelata is significantly lower in priced compared to the pileus. The aim of this study was to conduct a comprehensive comparative analysis of the nutritional and biological properties between the pileus and stipe of M. sextelata. The results revealed that the stipe exhibited comparable levels of various nutrients and bioactive compounds to those found in the pileus. The stipe showed significantly higher levels of crude dietary fiber, various mineral elements, vitamins, amino acids, 5′-nucleotides, fatty acids, and specific sugars. Additionally, it also demonstrated significant abundance in bioactive compounds such as total flavonoids and ergothioneine. Overall, our study provides valuable insights into unlocking further knowledge about M. sextelata’s nutritional composition while highlighting its potential health benefits associated with different parts of this highly esteemed edible mushroom.


Introduction
Morchella sextelata, commonly known as black morel mushroom, is a type of edible fungus that is highly sought after by consumers for its distinctive flavor, nutritional value and texture.This species has garnered widespread attention due to its culinary appeal, where it is regarded as a delicacy in many cultures (1).The nutritional composition and biological properties of this species have gained significant attention due to its potential applications in the culinary, pharmaceutical, and nutraceutical industries (2).It possesses the potential to emerge as a significant source of nutritional diets and bioactive compounds.However, little is known about the underlying nutritional composition and biological properties that differentiate the different parts of this intriguing organism.Therefore, further investigation is necessary to explore the distribution of nutrients and bioactive compounds in this morel mushroom, in order to satisfy the increasing demand for these valuable edible fungi and maximize their utilization.
M. sextelata is a medium to large-sized mushroom that can reach heights of up to 15-20 cm.It possesses a distinct cone-shaped cap that exhibits a dark brown to black hue, featuring deep pits and ridges which lend it an appearance of honeycomb (3).The pileus is The M. sextelata strain (CCMJ5600) was obtained from the Culture Collection Center of Mycology of Jilin Agriculture University.For pure culture isolation, a 5 mm punch from solid medium was inoculated onto the center of potato dextrose agar (PDA, Difco, Becton-Dickinson Co., Sparks, MD, United States) and incubated at 24°C for 5 days.The cultivation process was conducted in an artificial climate chamber following previously published methods (8).Mature fruiting bodies of M. sextelata were collected, and the pileus and stipe were separated (Figure 1).The pileus and stipe were freeze-dried using a vacuum freeze dryer (Christ ALPHA 1-2LD plus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).The freeze-dried pileus and stipe were subsequently ground into powder and stored at −80°C.The freeze-dried ground powder was used to determine all components except for moisture.

Determination of fat
The fat content in the pileus and stipe of M. sextelata was determined using a Soxhlet extraction apparatus, following a previously established method (9).The fat content was determined by equation (2):

Determination of the total ash
The ash content in the pileus and stipe was determined using a previously established method (10).The ash content was calculated as equation ( 3): w1: the weight of an empty crucible; w2: the weight of the crucible containing ash; w3: the weight of the crucible with pileus or stipe sample.

Determination of crude dietary fiber
A diverse array of Association of Official Analytical Chemists (AOAC) official methods of analysis have been developed and endorsed for the quantification of DF.DF and insoluble dietary fiber (IDF) analysis were determined using the method described by AOAC (11).The extraction and determination of soluble dietary fiber (SDF) in the pileus and stipe was performed using a previously established method (12).

Determination of crude protein
The crude protein contents were determined using the method described previously, with BSA being used as the standard (13).

Determination of the total carbohydrates
The total carbohydrate content was determined using the phenolsulfuric acid method (14).The absorbance was measured at 490 nm utilizing a UV-visible spectrophotometer (U-5100, HITACHI, Tokyo, Japan).The total carbohydrate content was determined by utilizing a standard curve derived from glucose.
Calorie content was calculated by adding the amount of calories obtained from carbohydrate, protein and fat.The amount of carbohydrate and protein in grams were multiplied by 4, while the amount of fat in grams was multiplied by 9.

Determination of mineral elements
The mineral elements content in the pileus and stipe of M. sextelata was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7850, Agilent Technologies, Inc., Santa Clara, CA, United States), including macro-elements (K, Ca, Na, Mg, P), trace elements (Fe, Zn, Se) and heavy metal elements (Mn, Cu) (15).The powder sample (0.25 g) was digested with 9 mL of 65% HNO 3 and 1 mL of 30% H 2 O 2 in a microwave digestion system (UltraClave MLS, Leutkirch, Germany) for 10 min, followed by dilution to a final volume of 50 mL with deionized water.

Determination of vitamins
In this study, various vitamins contained in the pileus and stipe were determined, including water-soluble vitamin (vitamin C, B 1 , B 2 , B 6 ) and fat-soluble vitamin (vitamin E, D 2 ).The vitamin C content was determined using high performance liquid chromatography (HPLC) as previous described in CNS GB 5009.86-2016 (18).The content of vitamin B 1 , B 2 , B 6 , D 2 , and E were determined using AOAC method (19).

Determination of fatty acids
Fatty acids were detected by MetWare 1 based on the Agilent 8,890-5977B gas chromatography mass spectrometry (GC-MS) platform.The specific sample preparation, extraction and detection method was referred to the method established by Tan et al. (20).The sample derivants were analyzed using an Agilent 8890-5977B GC-EI-MS system.

Determination of total sugars, sugar alcohols and sugar acids
The total sugar, sugar alcohol and sugar acid were detected by Novogene Co., Ltd.(Beijing, China) based on Agilent GC-MS platform.Briefly, 50 mg of the sample was mixed with 1 mL of 80% MeOH and left for 30 min at −20°C.The mixture was then centrifuged (15,000 g at 4°C for 15 min) and diluted 10 times with 20 μL supernatant.Subsequently, 20 μL of the diluent and 5 μL of internal standard (100 μg/mL Glucose-13 C 6 ) were dried in a GC-MS bottle.30 μL of methoxyamine hydrochloride pyridine was added into it and incubated at 37°C for 90 min.Then, 30 μL of BSTFA was added and incubated at 70°C for 60 min, cooled at room temperature.The final sample was injected into Agilent 7980A/5975C GC-MS system (Agilent Technologies, Inc., Santa Clara, CA, United States) with a column (MACHEREY-NAGEL, 30 m × 0.25 mm × 0.25 μm, Germany).

Distribution of bioactive compounds in the pileus and stipe 2.4.1 Extraction of crude polysaccharides
The crude polysaccharides in the pileus and stipe of M. sextelata were extracted using a previously established method with some modifications (21).Briefly, the powder sample (5 g) was mixed with water (200 mL) and subjected to heating at 90°C for a duration of 3 h.Subsequently, the supernatant was collected by centrifugation (Avanti JXN-26, Beckman Coulter Commercial Enterprise Co., Ltd., Bria, California, United States) at 12,000 rpm for 10 min.The result solution was mixed with equal volume chilled ethanol (96%) and kept at 4°C overnight.The precipitate was then collected by centrifuging at 1 http://www.metware.cn/15,000 rpm for 10 min, followed by two washes with 96% ethanol.Finally, the obtained precipitate was freeze dried in a vacuum freeze dryer.The crude polysaccharide content was calculated by equation ( 4): m1: the weight of crude polysaccharide; m2: the weight of initial sample.

Determination of the total polysaccharides
The total polysaccharide content in the crude polysaccharides extracted from both the pileus and stipe was quantified using the phenol-sulfuric acid method (22).The standard curve was constructed using glucose.

Determination of total phenolic
The total phenolic content in the pileus and stipe was quantified using a previously validated methodology, which employed Folin-Ciocalteu reagent and gallic acid as standard for measurement (23).

Determination of total flavonoid
The flavonoid contents in the pileus and stipe were determined as previously described ( 23), with quercetin being used as the reference standard.

Determination of total triterpenes
Total triterpenes content was determined by a previously established method with some modifications (24).Briefly, the powder sample (0.2 g) was added to 50 mL of anhydrous ethanol and thoroughly mixed using vortex agitation.The sample was subsequently subjected to ultrasonic extraction at 500 W for 1 h, followed by collection through centrifugation at 8,000 r/min for 10 min.The supernatant was dried at 90°C and mixed with vanillin-glacial acetic acid solution (100 μL, 5% w/v) and perchloric acid solution (800 μL).The sample solutions were heated at 60°C for 45 min and subsequently cooled to ambient temperature in an ice-water bath.The solution was supplemented with 5 mL of glacial acetic acid and incubated at room temperature for 10 min to measure the absorbance at 548 nm.The standard curve was constructed using ursolic acid.The fluorescence units of ursolic acid per unit weight were calculated and obtained to determine the values.

Determination of ergosterol
The HPLC method was utilized for the determination of ergosterol content in both the pileus and stipe, as described by Jasinghe and Perera (25).The final sample was injected into HPLC system (Agilent 1290) with a C18 column (YMC Pack ODS-AM-303, 4.6 mm × 250 mm, 5 μm; Agilent Technologies, Wilmington, DE, United States).

Determination of ergothioneine
The ergothioneine content in the pileus and stipe was quantified using the HPLC method established by Islam et al. (14).The extracted samples were analyzed using an HPLC system (Alliance system, Germany) equipped with a UV-DAD detector (Alliance 1200 Series, Germany).The analysis was carried out using an Agilent Eclipse

Statistical analysis
The analyses were conducted in triplicate, and the results were reported as mean ± standard deviation (SD).Significant differences (p < 0.05) among the samples were determined using one-way ANOVA with Duncan's multiple range tests.Statistical analysis was performed using SPSS software (version 19.5, IBM SPSS Statistics, Armonk, NY, United States).

Proximate composition of the pileus and stipe
The utilization of morel mushrooms for human consumption has long been established, owing to their renowned texture, flavor, and medicinal properties.Fruiting bodies of morel mushrooms typically exhibit a moisture content of approximately 90%.Their dry matter is rich in carbohydrates (50-65%) and proteins (19-35%), while containing minimal quantities of fats (2-6%) (7).Consequently, they are considered an ideal source of high-quality dietary food.In this study, the proximate composition of the pileus and stipe was quantified, including moisture, fat, crude dietary fiber, crude protein, total ash, and carbohydrate (Table 1).The moisture content of the fresh pileus was found to be higher than that of the fresh stipe.Therefore, the stipe is more suitable for preservation and long-distance transportation due to its lower moisture level, which helps to effectively inhibit microbial and enzyme activity.Additionally, morels are highly esteemed for their elevated protein content and minimal fat levels.As shown in Table 1, both the pileus and stipe contained exceptionally high protein levels, with no significant disparity between them, indicating their equivalent nutritional value as exceptional sources of wholesome protein.Furthermore, both the pileus and stipe demonstrated an exceptionally low fat content level (Table 1).Their fat content is even lower than that of previously reported morel mushrooms and some other edible mushrooms, such as shiitake mushrooms or oyster mushroom (7).Mushrooms are a low-calorie food.In this study, both the pileus and stipe showed lower energy values, with the energy value in the stipe being significantly lower than that in the pileus (Table 1).This further indicates that the stipe is an excellent source of low-calorie food.
The ash content in food products indicates the quantity of inorganic residue remaining as a result of the ashing process or hightemperature heating, and/or the degradation of organic components through strong acid treatment.In this study, variations were observed between the pileus and stipe regarding their ash content levels, with the stipe exhibiting a higher level (16.33%).Previous studies have identified variations in the ash content among different species of morels (26).Our findings demonstrate that M. sextelata exhibits comparable ash content to other morel species.
The health benefits of DF in mushrooms have been extensively demonstrated in numerous previous studies, encompassing its hypoglycemic effect, regulatory mechanism on pancreatic lipase, modulation of gut microbiota, and activation of macrophages (27).In this study, it was observed that the pileus part had a significantly higher content of DF compared to the stipe part.DF is typically classified into IDF and SDF, with IDF generally surpassing SDF in terms of content.By binding with glucose and inhibiting amylase, IDF can modulate the gut microbiota composition to prevent colitis, increase fecal volume, and reduce postprandial blood sugar levels, thereby achieving the preventive effect against diabetes (28).The significant health benefits associated with SDF have garnered considerable consumer interest, including reductions in serum cholesterol levels, improvements in postprandial blood sugar control, and lowered blood lipid levels.Mushrooms serve as abundant sources of SDF (27).However, it should be noted that the content of SDF in mushrooms can vary significantly depending on factors such as variety, growth stage, and different parts (27).As shown in Table 1, the pileus and stipe parts both contained SDF and IDF components.However, the stipe part exhibited significantly higher levels.The results suggest that the stipe part rich in DF content is also a highly recommended dietary option.
Carbohydrates play a crucial role in providing the essential energy for the formation and growth of morel fruiting bodies, which make up the majority of their dry weight.Mushrooms are abundant in both easily digestible carbohydrates (such as glucose, glycogen, mannitol, and trehalose) and indigestible carbohydrates (including chitin, β-glucan, and mannans) (7).There were no significant difference in carbohydrate content between the pileus and stipe (Table 1).The flavor of morel mushroom can be enhanced by soluble monosaccharides and sugar alcohols generated from the hydrolysis of stable carbohydrates.
Carbohydrates, proteins, and fats are macronutrients essential for the body's growth, energy provision, and maintenance of various physiological functions.According to the results of this study, the macronutrient composition of the pileus and stipe does not exhibit any significant disparity.Therefore, it can be concluded that both the pileus and stipe of M. sextelata are nutritionally sound as they are rich sources of carbohydrates, proteins, fibers while having low levels of fat content.In recent years, the market has witnessed a wide range of commercially available value-added mushroom products, encompassing sauces, seasonings, extracts, and mushroom supplements (29).The incorporation of sextelata into flour-based products such as bread and cookies, along with the integration of mushroom powder into various food items like muffins, breads, pasta, and snacks serves to augment their nutritional and biological profile.

Analysis of mineral elements and vitamins in the pileus and stipe
The mineral elements found in morel mushrooms are not only essential for various physiological functions in the human body, but they also make a significant contribution to their overall nutritional value.These minerals play crucial roles in various biological processes and are necessary for maintaining optimal health (30).In this study, we conducted a detailed analysis of the mineral element contents of both macro and trace elements, as well as certain heavy metal elements, in the pileus and stipe samples (Table 2).The results showed significant differences in the macro element content between the pileus and stipe, with higher levels observed in the pileus.Specifically, it was found that the pileus exhibited considerably higher levels of Fe and Zn, while Se was found to be rich in the stipe.These trace elements are known to participate in vital biological processes such as enzyme activation, regulation of immune system function, and antioxidant defense mechanisms (21).Interestingly, compared to other types of morel mushrooms, M. sextelata demonstrated comparatively higher levels of trace elements such as Fe, Zn, Se (7).This suggests that consuming M. sextelata may provide greater nutritional benefits due to its elevated mineral concentrations.Additionally, the pileus and stipe samples of M. sextelata show lower levels of certain heavy metal elements (Mn: 6.10-9.02mg/Kg; Cu: 6.86-9.42mg/Kg) compared to other morel mushrooms (23).Overall, understanding the mineral element contents of morel mushrooms like M. sextelata provides insights into their nutritional value and can aid individuals who seek a balanced diet or researchers studying food composition for dietary recommendations.
In addition to these important minerals, morel mushrooms also provide significant amounts of other beneficial nutrients such as vitamins (7).Vitamins play a crucial role in maintaining various bodily functions.Morel mushrooms are particularly rich in vitamin B such as vitamin B 2 , niacin (B 3 ), pantothenic acid (B 5 ), and folate (B 9 ) (7).As shown in Table 2, the levels of different vitamins were measured in both the pileus and stipe.The pileus and stipe of M. sextelata exhibited remarkably high concentrations of vitamin B 1 and vitamin C, with no significant difference in vitamin C content between the two parts.Both vitamin B 1 and vitamin C play vital roles in maintaining optimal health.The presence of vitamin B 1 ensures efficient energy production from food sources to meet the body's metabolic demands, while vitamin C facilitates proper digestion processes and supports optimal functioning of the nervous system.Incorporating these essential vitamins into a well-balanced diet can significantly contribute to overall well-being (5).However, the stipe exhibited significantly higher levels of vitamin B 1 , B 2 , and D 2 content compared to the pileus; conversely, it showed significantly lower levels of vitamin B 6 and E.
By comprehending the mineral profiles of the pileus and stipe in M. sextelata, our research demonstrates that both the stipe and pileus serve as exceptional sources of essential mineral elements and dietary supplements.The stipe of M. sextelata is considered to be a significant source of vital nutrients.The utilization of the stipe has the potential to effectively alleviate nutritional deficiencies commonly found in impoverished and malnourished populations.

Analysis of the amino acid and 5′-nucleotides in the pileus and stipe
The consumption of mushrooms, which are abundant in amino acids and nucleotides, not only provides a delectable culinary experience but also offers exceptional nutritional value due to their prominent flavor characteristics and unique texture.Amino acids and nucleotides are known to contribute to flavor enhancement in food products because of their umami taste properties.The umami taste, also known as the savory sensation, is derived from amino acid compounds such as Glu and Asp., as well as flavor nucleotides like 5′-IMP and 5′-GMP (31).In this study, eight essential and nine non-essential amino acids were identified and quantified in the pileus and stipe.As shown in Table 3, the pileus and stipe contained a comprehensive range of amino acids that encompassed all the tested varieties.The essential amino acids constituted over 40% of the total amino acid content, while the ratio of essential to non-essential amino acids (E/N value) exceeded 0.6, thereby satisfying the ideal protein condition proposed by FAO/WHO (32).The pileus had a significantly higher total amino acid content compared to the stipe (Table 3).However, both exhibited remarkably high levels of total amino acids, indicating their exceptional nutritional value.Moreover, both the pileus and stipe of M. sextelata showed a high abundance of Glu and Asp., which aligns with previous findings in other mushrooms (16).The amino acid Glu (associated with umami taste) was found to be the most predominant in both the pileus and stipe, whereas the content Values are reported as means ± SD of three determinations.Those with different letters in the same row showed significant differences among the groups (p < 0.05).Values are reported as means ± SD of three determinations.
of Asp (also associated with umami taste) in the pileus was significantly higher compared to that in the stipe.The results suggest that Glu and Asp may play significant roles as the key amino acids influencing the flavor profile of M. sextelata.However, both the pileus and stipe exhibited significantly elevated levels of these two amino acids, resulting in a pronounced umami flavor profile.Simultaneously, the pileus and stipe also contained abundant amino acids that contributed to a sweet taste sensation.The content of these delectable amino acids in M. sextelata surpassed that of previously reported edible mushrooms such as Flammulina velutipes, shiitake mushrooms, oyster mushrooms, etc., thereby contributing to its exquisite taste and widespread popularity (33).
Furthermore, precise quantification of 5′-nucleotides was achieved within both the pileus and stipe.The inclusion of key nucleotides such as 5′-IMP, 5′-GMP, 5′-AMP, 5′-CMP, and 5′-UMP provided a detailed understanding of their distribution throughout the fruiting body.Based on quantitative analysis, the total content of these 5′-nucleotides in the stipe was significantly higher than that in the pileus.The main 5′-nucleotides responsible for umami taste are 5′-IMP and 5′-GMP (17).Notably, the concentration of 5′-GMP was higher in the entire fruiting body of morels with a significantly greater amount found in the stipe compared to the pileus.Identifying specific types and quantities of amino acids and 5′-nucleotides present in the fruiting body contributes to enhancing culinary applications or potentially uncovering health benefits associated with consuming M. sextelata.The neglected stipe part can be utilized more effectively and suitable condiments of mushroom sauce can be developed based on their nutritional and flavor characteristics.

Fatty acid profiles in the pileus and stipe
A total of 33 fatty acids were detected in M. sextelata, and their percentage contents in the total fatty acids and specific contents in the pileus and stipe were shown in Table 4 and Supplementary Table S1, respectively.Among these, six were classified as medium chain fatty acids (decanoic acid, nonanoic acid, octanoic acid, hexanoic acid, hendecanoic acid and lauric acid) while the remaining 27 belonged to the long-chain category.Additionally, 16 were classified as unsaturated fatty acids, while the remaining 17 were categorized as saturated fatty acids (SFA).The total fatty acid content in the stipe was significantly higher than that in the pileus.Previous studies have found that the fat fraction in mushrooms primarily consists of unsaturated fatty acids (2).It is noteworthy that the amount of unsaturated fatty acids exceeds that of saturated fatty acids, which is consistent with findings from previous studies conducted on similar species (7).Furthermore, the stipe contains higher levels of polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA) compared to the cap, which further indicates that the stipe is a nutritious food source.In this study, it was found that linoleic acid, which belongs to unsaturated fatty acids, had the highest content among all fatty acids in both the pileus and stipe, followed by palmitic acid and oleic acid.The linoleic acid serves as a precursor for the formation of 1-octen-3-ol, an essential aromatic compound responsible for imparting the distinctive flavor profile found in mushrooms (34).Additionally, linoleic acid is an essential fatty acid that cannot be synthesized in the human body.It can produce γ-linolenic acid and ultimately generate prostaglandins, which regulate various physiological processes such as blood pressure, heart vessel protection, and prevention of arteriosclerosis.The composition of fatty acids can be used to evaluate their nutritional index and explore their potential applications in disease prevention and treatment (35).The ratio of PUFA/SFA is an index commonly used to evaluate the impact of diet on cardiovascular health (CVH).It assumes that all PUFAs in the diet can reduce low-density lipoprotein cholesterol (LDL-C) and serum cholesterol levels, while all SFAs contribute to an increase in serum cholesterol levels.Therefore, the higher the ratio, the more positive the effect.The cap and stipe ratio of six sister morel reached 3.03 and 2.93 respectively, which is higher than common meat, and fish ratios.The Index of Atherogenicity (IA) has also been widely used to evaluate seaweed, crops, meat, fish, dairy products etc.The IA ratio of Morchella esculenta cap and stipe falls within the same range.The Health-promoting Index (HPI) is calculated as the reciprocal of IA and is currently mainly used for research on dairy products with values ranging from 0.16 to 0.68.Dairy products with high HPI values are considered more beneficial for human health.The HPI values of M. sextelata pileus and stipe are much higher than those of dairy products indicating that M. sextelata pileus and stipe have a greater potential for promoting human health in terms of fatty acid ratio compared to dairy products.The identification and quantification of these fatty acids provide valuable insights into the nutritional value and potential health benefits associated with consuming M. sextelata.Overall, this study illuminates the remarkable diversity and complexity within M. sextelata's lipid profile by identifying 33 different fatty acids present in both its pileus and stipe.These findings deepen our understanding of this fascinating fungus while also opening up avenues for future exploration regarding its culinary uses or potential therapeutic applications.

Analysis of total sugars, sugar alcohols and sugar acids
As shown in Table 5, both the pileus and stipe of M. sextelata exhibited a wide range of sugars, sugar alcohols, and sugar acids.The stipe showed significantly elevated levels for the majority of detected sugars, sugar alcohols, and sugar acids.The presence of these compounds indicates the nutritional value and potential health benefits associated with consuming both the stipe and pileus of M. sextelata.Among the various types of sugars detected, there were seven monosaccharides identified which include glucose, fructose, galactose, rhamnose, mannose, and fucose (Table 5).These monosaccharides are essential for energy production in our bodies and play important roles in various metabolic processes.In addition to monosaccharides, trehalose was identified as the only disaccharide present in M. sextelata.Trehalose is renowned for its ability to the flavor of food while also serving as an energy source.The fact that M. sextelata has a higher content of trehalose compared to other edible mushrooms implies that it could be an exceptional option for individuals in search of a natural sweetener or desiring to incorporate sweetness into their culinary creations (36).Meanwhile, the stipe, with its higher concentration of trehalose, serves as an excellent reservoir for this valuable sugar.Additionally, both parts of M. sextelata contained six different sugar alcohols: xylitol, mannitol, sorbitol, arabitol, fucitol and myo-inositol.Sugar alcohols are commonly used as low-calorie alternatives to traditional sugars due to their reduced impact on blood sugar levels.Lastly but not least importantly glucuronic acid and gluconic acid were detected as two sugar acids present in this mushroom species.These organic acids have been linked to numerous health benefits, such as antioxidant properties and anti-inflammatory effects.Overall, the comprehensive analysis conducted on the pileus and stipe of M. sextelata highlights its rich composition of sugars, sugar alcohols, and sugar acids that contribute to its nutritional value.

Analysis of polysaccharide
Firstly, the crude polysaccharide content of both the pileus and stipe in M. sextelata was thoroughly investigated.The extracted crude polysaccharides from the pileus and stipe showed no significant difference (Table 6).The pileus had a crude polysaccharide content of 73.10 mg/g, while the stipe exhibited a similarly high content of 69.96 mg/g.The crude polysaccharide is composed of a combination of polysaccharides with different molecular weights, exhibiting diverse compositions and functions (37).There was no significant difference in the total polysaccharide content between the pileus and stipe.Polysaccharides in mushrooms have various activities, such as antibacterial, anti-tumor, anti-fatigue effects, and the ability to inhibit the proliferation of cancer cells (4).Due to its low price, the stipe of M. sextelata can be used as an economical raw material for extracting Morchella polysaccharide.With its broad application prospects and market demand, it also has great potential as a raw material for natural medicine and health products.

Analysis of total phenolic and flavonoid compounds
The phenolics, a group of chemical compounds found in mushrooms, play a crucial role in providing antioxidant activity (14).Phenolic and flavonoids compounds are the predominant phenolics in morel mushrooms (30).As shown in Table 6, the pileus exhibited significantly higher levels of total phenolic content, while the stipe demonstrated notably elevated levels of flavonoids.The phenolic content in both the pileus and stipe of M. sextelata was relatively lower compared to previously reported morel mushrooms (23).The flavonoid contents generally exhibited significantly lower levels compared to the phenolics.However, the total flavonoid contents in its pileus and stipe is higher than that reported for many other morel mushrooms (7).In conclusion, due to its rich content of phenolics and higher flavonoids with significant biological activities, the stipe holds great promise for further research into its potential uses across multiple industries aimed at improving human health outcomes through innovative products development.

Analysis of the total triterpenes
As shown in Table 6, the pileus was found to have a total triterpenes content of approximately 23.06 mg/g, while the stipe exhibited a slightly lower concentration of around 19 mg/g.Statistical analysis revealed no significant difference (p > 0.05) in the total triterpenes contents between the pileus and stipe.Triterpenes are known for their diverse biological activities and potential therapeutic applications, which makes them an important focus of research in various fields such as medicine and pharmacology (24).The results illuminates the distribution pattern of triterpenes within different parts of M. sextelata, laying a foundation for future investigations aimed at harnessing its medicinal or industrial potentials.

Analysis of ergosterol
Ergosterols are important precursors of vitamin D 2 and are the main sterols found in morels (7).Previous studies have demonstrated that ergosterol possesses remarkable antioxidant and antimicrobial properties, without causing any hepatotoxic effects (38).In this study, the presence of substantial quantities of ergosterol were observed in both the pileus and stipe (Table 6).The pileus exhibited higher ergosterol contents (approximately 2.87 mg/g), whereas the stipe displayed lower content (approximately 2.70 mg/g).However, compared to some previously reported staple mushrooms such as Auricularia auricula and oyster mushroom (14, 23), the content of ergosterol in the pileus and stipe of M. sextelata is higher.Furthermore, these high concentrations of ergosterol also contribute to the extensive synthesis of vitamin D 2 in M. sextelata, thereby further enhancing its nutritional value.

Analysis of ergothioneine
Mushrooms are the richest dietary source for ergothioneine (39).As a natural antioxidant, ergothioneine has various physiological Values are reported as means ± SD of three determinations.Those with different letters in the same row indicated significant differences between the pileus and stipe within the same row at p < 0.05.Values are reported as means ± SD of three determinations.functions such as scavenging free radicals, detoxification, maintaining DNA biosynthesis and cell growth, and enhancing immunity.In this study, the ergothioneine content in the stipe was significantly greater than that in the pileus (Table 6).This suggests that the stipe's high concentration of ergothioneine may provide it with excellent biological activity comparable to that of the pileus.

Conclusion
Our work presents the first comparative analysis of nutritional and biological properties between the pileus and stipe of M. sextelata, a highly esteemed edible mushroom.The stipe exhibited nutrients and bioactive ingredients that were not inferior to the pileus, such as crude DF, various minerals and vitamins, several amino acids and 5′-nucleotides, total flavonoid, and ergothioneine.Our findings shed light on the potential variations in the nutritional composition and potential benefits associated with different parts of this fungus.The results will contribute to exploring the bioavailability of compounds as well as their potential synergistic effects in relation to human health.

FIGURE 1
FIGURE 1 The fruiting body of M. sextelata.(A) A model of the fruiting body of M. sextelata.(B) The pileus part of a mature fruiting body.(C) The stipe part of a mature fruiting body.The powder is ground from freeze-dried parts of stipe (D) and pileus (E).

TABLE 1
Proximate composition of the pileus and stipe.
bValues are reported as means ± SD of three determinations.The groups denoted by different letters indicate a significant difference at p < 0.05.Values with no superscript are not statistically significant.

TABLE 2
The content of mineral elements and vitamins in the pileus and stipe of M. sextelata.

TABLE 3
Comparison of the amino acids and 5′-nucleotides contents between the pileus and stipe.The total amount of essential amino acids; N: The total amount of non-essential amino acids; S: Total sweet amino acids; F: Total umami amino acids = Asp + Glu; T: Total amino acid.Values are reported as means ± SD of three determinations.Those with different letters in the same row showed significant differences among the groups (p < 0.05).Values are reported as means ± SD of three determinations. E:

TABLE 4
Composition of fatty acids in the pileus and stipe (dry weight basis, % of total fatty acid).

TABLE 5
Comparison of total sugars, sugar alcohols and sugar acids in the pileus and stipe.Values are reported as means ± SD of three determinations.Those with different letters in the same row showed significant differences among the groups (p < 0.05).Values are reported as means ± SD of three determinations.

TABLE 6
Comparison of bioactive compounds between the pileus and stipe.